Bone marrow mesenchymal stem cells (MSCs) have tremendous potential as a non-immunogenic or autologous cell source for tissue regeneration. However, how the differentiation and function of MSCs are regulated by mechanical and chemical factors, especially in three-dimensional (3D) matrix, is not well understood. The long-term goal of this project is to use adult stem cells to construct tissues (e.g., vascular grafts) for cardiovascular repair. In this project, we propose to use tissue-engineered vascular grafts as a model to investigate the differentiation and function of MSCs in response to vascular mechanical and chemical stimuli in 3D matrix. When using stem cells to construct tissue-engineered vascular grafts, cell differentiation and matrix remodeling are two major issues. Based on our preliminary studies, we hypothesize that cyclic mechanical strain promotes MSC differentiation into vascular smooth muscle cells (SMCs) and enhance matrix production in 3D vascular graft constructs, which is synergized by transforming growth factor beta (TGF- beta). The objectives of this project are: (1) To investigate the differentiation of MSCs into SMCs in response to mechanical strain and/or TGF-beta in 3D matrix;(2) To investigate the matrix remodeling by MSCs in response to mechanical strain and/or TGF-beta in 3D matrix. To investigate the mechanical and chemical regulation of MSC differentiation, we will culture MSCs in vascular graft constructs made of a collagen-fibrin gel matrix. To examine the effects of TGF- beta, we will include TGF-beta in some grafts. We will use a bioreactor to generate cyclic strain to mimic the mechanical environment of a blood vessel. DMA microarrays will be used to profile the genetic re-programming of MSCs. To determine matrix remodelling in our vascular grafts, the production of matrix proteins and the activity of metalloproteinases will be quantified. The mechanical properties of the vascular grafts will be measured using an Instron Test Machine. This research will break new grounds on stem cell engineering and tissue engineering, and advance our understanding on the roles of mechanical and chemical factors in the differentiation and function of MSCs. The results will provide a rational basis and novel approach for vascular tissue engineering that will eventually benefit our society. For example, this research will make it possible to use patients'bone marrow to construct autologous and non-immunogenic blood vessels. This research will also contribute to the knowledge of mechanobiology of stem cells in 3D matrix, which is critical but not well understood.